The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis

Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.     

Spinal N-methyl-D-aspartate receptors and nociception-evoked release of primary afferent substance P

Dorsal horn N-methyl-D-aspartate (NMDA) receptors contribute significantly to spinal nociceptive processing through an effect postsynaptic to non-primary glutamatergic axons, and perhaps presynaptic to the primary afferent terminals. The present study sought to examine the regulatory effects of NMDA receptors on primary afferent release of substance P (SP), as measured by neurokinin 1 receptor (NK1r) internalization in the spinal dorsal horn of rats. The effects of intrathecal NMDA alone or in combination with D-serine (a glycine site agonist) were initially examined on basal levels of NK1r internalization. NMDA alone or when co-administered with D-serine failed to induce NK1r internalization, whereas activation of spinal TRPV1 receptors by capsaicin resulted in a notable NK1r internalization. To determine whether NMDA receptor activation could potentiate NK1r internalization or pain behavior induced by a peripheral noxious stimulus, intrathecal NMDA was given prior to an intraplantar injection of formalin. NMDA did not alter the formalin-induced NK1r internalization nor did it enhance the formalin paw flinching behavior. To further characterize the effects of presynaptic NMDA receptors, the NMDA antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 were intrathecally administered to assess their regulatory effects on formalin-induced NK1r internalization and pain behavior. AP-5 had no effect on formalin-induced NK1r internalization, whereas MK-801 produced only a modest reduction. Both antagonists, however, reduced the formalin paw flinching behavior. In subsequent in vitro experiments, perfusion of NMDA in spinal cord slice preparations did not evoke basal release of SP or calcitonin gene-related peptide (CGRP). Likewise, perfusion of NMDA did not enhance capsaicin-evoked release of the two peptides. These results suggest that presynaptic NMDA receptors in the spinal cord play little if any role on the primary afferent release of SP.     

BDNF improves the effects of neural stem cells on the rat model of Alzheimer's disease with unilateral lesion of fimbria-fornix

In this study, neural stem cells (NSCs) were obtained from the hippocampus using the serum-free culturing. NSCs labeled with 5'-bromo-2'-deoxyuridine (BrdU) were transplanted into transected rat basal forebrain followed by the injection of brain-derived neurotrophic factor (BDNF) into the lateral ventricle. Nestin staining and double-labeling immunohistochemistry were used to detect cell survival and neuronal differentiation of the BrdU labeled cells in the basal forebrain and it was observed that labeled NSCs differentiated into neurons and astrocytes in the basal forebrain. Immunohistochemical detection of p75(NGFR) indicated that the number of cholinergic neurons of the combination groups treated by NSCs, BDNF, and NSCs groups had more significant improvement than that of the injured groups in medial septum (MS) and vertical diagonal branch (VDB). Learning and memory abilities were also measured by Y-maze test and the results support that BDNF can enhance the treatment effects of NSCs transplanted into brain lesion model.     

Assessment of diagnostic accuracy and feasibility of dynamic telepathology in China

To assess the feasibility, including diagnostic accuracy and time cost, of a real-time telepathology system with pathologic slides, 600 cases covering a wide spectrum of lesions from 16 organ systems were tested. The "correct" diagnosis (gold standard) was established as a consensus by 2 experienced pathologists. The cases were first examined by 4 pathologists at different levels of experience with dynamic telepathology. Cases were then reviewed by the same pathologists using light microscopy in a blinded fashion 3 weeks to 2 months later. A diagnosis, together with reading times for telepathology and light microscopy, was recorded for each case. Diagnostic accuracy by telepathology was 94.8% (569/600), 93.3% (560/600), 91.6% (550/600), and 97% (388/400) for pathologists A, B, C, and D, respectively. Telepathologic diagnosis was concordant with the gold standard and with direct microscopy, with a mean of 94.2% and 99.26%, respectively. Most cases (510 or 85%) were diagnosed in 15 to 40 minutes by telepathology, with a mean of 17.0 minutes. The time needed to review a slide by telepathology was 3 to 4 times longer than that of standard light microscopy. All 4 pathologists were able to render a diagnosis in all cases. Our results showed that robotic telepathology is sufficiently accurate for primary diagnosis in surgical pathology, but modifications in laboratory protocols, telepathology hardware, and internet speed are needed to reduce the time necessary for diagnosis by telepathology before this method may be deemed suitable for use in a busy practice.     

Translation of ASH1 mRNA is repressed by Puf6p-Fun12p/eIF5B interaction and released by CK2 phosphorylation

Translational repression during mRNA transport is essential for spatial restriction of protein production. In the yeast Saccharomyces cerevisae, silencing of ASH1 mRNA before it is localized to the bud cortex in late anaphase is critical for asymmetric segregation of Ash1p to the daughter cell nucleus. Puf6p, an ASH1 mRNA-binding protein, has been implicated in this process as a translational repressor, but the underlying mechanism is unknown. Here, we used yeast extract-based in vitro translation assays, which recapitulate translation and phosphorylation, to characterize the mechanism of Puf6p-mediated translational regulation. We report that Puf6p interferes with the conversion of the 48S complex to the 80S complex during initiation, and this repression by Puf6p is mediated through the general translation factor eIF5B (Fun12p in S. cerevisiae). Puf6p interacts with Fun12p via the PUF domain, and this interaction is RNA-dependent and essential for translational repression by Puf6p. This repression is relieved by phosphorylation of the N-terminal region of Puf6p mediated by protein kinase CK2 (casein kinase II). Inhibition of phosphorylation at Ser31, Ser34, and Ser35 of Puf6p increases its translational repression and results in ASH1 mRNA delocalization. Our results indicate that Puf6p suppresses the translation initiation of ASH1 mRNA via interaction with Fun12p during its transport, and this repression can be released by CK2 phosphorylation in the N-terminal region of Puf6p when the mRNA reaches the bud tip.     

Functional morphology and physiological properties of bronchopulmonary C-fiber afferents

Nonmyelinated (C-) fibers represent the majority of vagal afferents innervating the airways and lung, and play an important role in regulating the respiratory and cardiovascular functions under both normal and abnormal physiologic conditions. Studies of the relationship between the conduction velocities of the vagal afferents and their sensitivities to capsaicin and other chemical irritants reveal that C-fibers are the primary type of chemosensitive afferents in the rat lung. Furthermore, a distinct sensitivity to capsaicin and a weak response to lung inflation are the defining characteristics of these afferents. In cultured rat nodose and jugular ganglion neurons, capsaicin-sensitive cells were identified by measurement of the capsaicin-evoked calcium transients using the Fura-2-based ratiometric imaging technique. The percentage of capsaicin-sensitive neurons gradually decreases as the cell diameter increases. However, the capsaicin-sensitive neurons cannot be precisely identified solely on the basis of the cell size. Anandamide, an endogenous cannabinoid released from leukocytes and epithelial cells, consistently evokes a stimulatory effect on pulmonary C-fiber endings by activating vanilloid receptor type 1 (VR1). The discharge pattern of pulmonary C-fibers evoked by anandamide closely resembles that produced by a much lower ( approximately 1/600) dose of capsaicin in the same fibers. Whether anandamide acts as a potential endogenous ligand to VR1 at the C-fiber terminals is unclear, and the physiological role of VR1 in modulating the transduction properties of these afferents also remains to be determined.     

Multiple envelope proteins are involved in white spot syndrome virus (WSSV) infection in crayfish

Summary. White spot syndrome virus (WSSV) is a devastating viral pathogen of cultured shrimp worldwide. Previous studies have shown that the intact virion consists of at least 39 structural proteins and, among them, six were identified as envelope proteins involved in the virus infection. In this paper, the structural proteins VP36A, VP36B and VP31 (J Virol 2004; 78: 11360–11370), containing the RGD motif, were expressed in Escherichia coli and used to produce specific antibodies. Western blot confirmed that VP36A is a newly reported envelope protein. A neutralization assay with these three antibodies demonstrated that VP36A, VP36B and VP31 could significantly delay the initial infection of crayfish, but mortality still reached 100% at day 11 post-injection. However, a neutralization assay with the combination of antibodies against different envelope proteins showed that a combination of VP36B and VP31 antibodies could strongly inhibit WSSV infection in crayfish. These results revealed that multiple envelope proteins are involved in WSSV infection in crayfish and that VP36B and VP31 play a key role during this process.     

Small peptide analogue of SDF-1alpha supports survival of cord blood CD34+ cells in synergy with other cytokines and enhances their ex vivo expansion and engraftment into nonobese diabetic/severe combined immunodeficient mice

The SDF-1/CXCR4 axis has been implicated in the chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. We studied the effects of a SDF-1 peptide analogue CTCE-0214 on the survival of cord blood CD34+ cells in culture, expansion, and engraftment of expanded cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Our results demonstrated that CTCE-0214 synergized with thrombopoietin (TPO), stem cell factor (SCF), or flt-3 ligand (FL) on the survival of stem and progenitor cells in culture. Adding CTCE-0214 at a low concentration (0.01 ng/ml) for 4 days together with TPO, SCF, and FL significantly enhanced ex vivo expansion of CD34+ cells to subsets of primitive (CD34+CD38- cells, colony-forming unit-mixed [CFU-GEMMs]), erythroid (CFU-Es), myeloid (CFU-GMs), and megakaryocytic (CD61+CD41+ cells, CFU-MKs) progenitors, as well as their multilineage engraftment in NOD/SCID mice. Interestingly, the short exposure of expanded cells to CTCE-0214 (100 and 500 ng/ml) for 4 hours did not increase the quantity of progenitor cells but enhanced their engraftment capacity. The proportion of CD34+ cells expressing surface CXCR4 was decreased, but the overall number of this population increased upon expansion. The small peptide analogue of SDF-1 could be developed for ex vivo expansion and improving engraftment of cord blood transplantation.     

Expression and functional analysis of Tgif during mouse midline development.

The Tgif gene encodes a homeodomain protein that functions as a transforming growth factor beta (TGF-beta) repressor by binding to Smad2. Mutations in the TGIF gene are associated with human holoprosencephaly, a common birth defect caused by the failure of anterior ventral midline formation. However, Smad2-mediated TGF-beta signaling in the axial mesendoderm has been demonstrated to be essential for ventral midline formation, and loss of a Smad2 antagonist should in principle promote rather than inhibit ventral midline formation. This suggests a more complex mechanism for the function of TGIF in controlling ventral midline formation. To explore the role of TGIF in ventral forebrain formation and patterning, we investigated Tgif expression and function during mouse development by in situ hybridization and gene targeting. We found that Tgif is highly expressed in the anterior neural plate, consistent with the proposed neural differentiation model in which TGF-beta suppression is required for normal neural differentiation. This result suggests a possible role for Tgif in anterior neural differentiation and patterning. However, targeted disruption of the Tgif gene during mouse development does not cause any detectable defects in development and growth. Both histological examination and gene expression analysis showed that Tgif-/- embryos have a normal ventral specification in the central nervous system, including the forebrain region. One interpretation of these results is that the loss of TGIF function is compensated by other TGF-beta antagonists such as c-Ski and SnoN during vertebrate anterior neural development.     

CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer's patches

T cell activation by dendritic cells (DCs) is critical to the initiation of adaptive immune responses and protection against pathogens. Here, we demonstrate that a specialized DC subset in Peyer's patches (PPs) mediates the rapid activation of pathogen specific T cells. This DC subset is characterized by the expression of the chemokine receptor CCR6 and is found only in PPs. CCR6(+) DCs were recruited into the dome regions of PPs upon invasion of the follicle associated epithelium (FAE) by an enteric pathogen and were responsible for the rapid local activation of pathogen-specific T cells. CCR6-deficient DCs were unable to respond to bacterial invasion of PPs and failed to initiate T cell activation, resulting in reduced defense against oral infection. Thus, CCR6-dependent regulation of DCs is responsible for localized T cell dependent defense against entero-invasive pathogens.